Kinase inhibitors for the treatment of disease

ABSTRACT

The present invention relates to organic molecules capable of modulating tyrosine kinase signal transduction in order to regulate, modulate and/or inhibit abnormal cell poliferation.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 10/306,975, filed Nov. 27, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to prodrugs of compounds capable ofmodulating, regulating and/or inhibiting tyrosine kinase signaltransduction. The present invention is also directed to methods ofregulating, modulating or inhibiting tyrosine kinases, whether of thereceptor or non-receptor class, for the prevention and/or treatment ofdisorders related to unregulated tyrosine kinase signal transduction,including cell growth, metabolic, and blood vessel proliferativedisorders.

2. Description of the Related Art

Protein tyrosine kinases (PTKs) comprise a large and diverse class ofproteins having enzymatic activity. The PTKs play an important role inthe control of cell growth and differentiation.

For example, receptor tyrosine kinase mediated signal transduction isinitiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic homeostasis, and responses tothe extracellular microenvironment).

With respect to receptor tyrosine kinases, it has been shown also thattyrosine phosphorylation sites function as high-affinity binding sitesfor SH2 (src homology) domains of signaling molecules. Severalintracellular substrate proteins that associate with receptor tyrosinekinases (RTKs) have been identified. They may be divided into twoprincipal groups: (1) substrates which have a catalytic domain; and (2)substrates which lack such domain but serve as adapters and associatewith catalytically active molecules. The specificity of the interactionsbetween receptors or proteins and SH2 domains of their substrates isdetermined by the amino acid residues immediately surrounding thephosphorylated tyrosine residue. Differences in the binding affinitiesbetween SH2 domains and the amino acid sequences surrounding thephosphotyrosine residues on particular receptors are consistent with theobserved differences in their substrate phosphorylation profiles. Theseobservations suggest that the function of each receptor tyrosine kinaseis determined not only by its pattern of expression and ligandavailability but also by the array of downstream signal transductionpathways that are activated by a particular receptor. Thus,phosphorylation provides an important regulatory step which determinesthe selectivity of signaling pathways recruited by specific growthfactor receptors, as well as differentiation factor receptors.

Aberrant expression or mutations in the PTKs have been shown to lead toeither uncontrolled cell proliferation (e.g. malignant tumor growth) orto defects in key developmental processes. Consequently, the biomedicalcommunity has expended significant resources to discover the specificbiological role of members of the PTK family, their function indifferentiation processes, their involvement in tumorigenesis and inother diseases, the biochemical mechanisms underlying their signaltransduction pathways activated upon ligand stimulation and thedevelopment of novel drugs.

Tyrosine kinases can be of the receptor-type (having extracellular,transmembrane and intracellular domains) or the non-receptor type (beingwholly intracellular).

The RTKs comprise a large family of transmembrane receptors with diversebiological activities. The intrinsic function of RTKs is activated uponligand binding, which results in phophorylation of the receptor andmultiple cellular substrates, and subsequently in a variety of cellularresponses.

At present, at least nineteen (19) distinct RTK subfamilies have beenidentified. One RTK subfamily, designated the HER subfamily, is believedto be comprised of EGFR, HER2, HER3 and HER4. Ligands to the Hersubfamily of receptors include epithelial growth factor (EGF), TGF-α,amphireguilin, HB-EGF, betacellulin and heregulin.

A second family of RTKs, designated the insulin subfamily, is comprisedof the INS-R, the IGF-1R and the IR-R. A third family, the “PDGF”subfamily includes the PDGF α and β receptors, CSFIR, c-kit and FLK-II.Another subfamily of RTKs, identified as the FLK family, is believed tobe comprised of the Kinase insert Domain-Receptor fetal liver kinase-1(KDR/FLK-1), the fetal liver kinase 4 (FLK-4) and the fms-like tyrosinekinase 1 (fit-1). Each of these receptors was initially believed to bereceptors for hematopoietic growth factors. Two other subfamilies ofRTKs have been designated as the FGF receptor family (FGFR1, FGFR2,FGFR3 and FGFR4) and the Met subfamily (c-met and Ron).

Because of the similarities between the PDGF and FLK subfamilies, thetwo subfamilies are often considered together. The known RTK subfamiliesare identified in Plowman et al, 1994, DN&P 7(6): 334-339, which isincorporated herein by reference.

The non-receptor tyrosine kinases represent a collection of cellularenzymes which lack extracellular and transmembrane sequences. Atpresent, over twenty-four individual non-receptor tyrosine kinases,comprising eleven (11) subfamilies (Src, Frk, Btk, Csk, Abl, Zap70,Fes/Fps, Fak, Jak, Ack and LIMK) have been identified. At present, theSrc subfamily of non-receptortyrosine kinases is comprised of thelargest number of PTKs and include Src, Yes, Fyn, Lyn, Lck, Blk, Hck,Fgr and Yrk. The Src subfamily of enzymes has been linked tooncogenesis. A more detailed discussion of non-receptor tyrosine kinasesis provided in Bolen, 1993, Oncogen 8: 2025-2031, which is incorporatedherein by reference.

Many of the tyrosine kinases, whether an RTK or non-receptor tyrosinekinase, have been found to be involved in cellular signaling pathwaysleading to cellular signal cascades leading to pathogenic conditions,including cancer, psoriasis and hyper immune response.

In view of the surmised importance of PTKs to the control, regulationand modulation of cell proliferation the diseases and disordersassociated with abnormal cell proliferation, many attempts have beenmade to identify receptor and non-receptor tyrosine kinase “inhibitors”using a variety of approaches, including the use of mutant ligands (U.S.Pat. No. 4,966,849), soluble receptors and antibodies (PCT ApplicationNo. WO 94/10202; Kendall & Thomas, 1994, Proc. Nat'l Acad. Sci 90:10705-09; Kim, et al, 1993, Nature 362: 841-844), RNA ligands (Jellinek,et al, Biochemistry 33: 10450-56); Takano, et al, 1993, Mol. Bio. Cell4:358A; Kinsella, et al, 1992, Exp. Cell Res. 199: 56-62; Wright, et al,1992, J. Cellular Phys. 152: 448-57) and tyrosine kinase inhibitors (PCTApplication Nos. WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808;U.S. Pat. No. 5,330,992; Mariani, et al, 1994, Proc. Am. Assoc. CancerRes. 35: 2268).

More recently, attempts have been made to identify small molecules whichact as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclicor heterocyclic aryl compounds (PCT Application No. WO 92/20642),vinylene-azaindole derivatives (PCT Application No. WO 94/14808) and1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have beendescribed generally as tyrosine kinase inhibitors. Styryl compounds(U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S.Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No.0 566 266 A1), seleoindoles and selenides (PCT Application No. WO94/03427), tricyclic polyhydroxylic compounds (PCT Application No. WO92/21660) and benzylphosphonic acid compounds (PCT Application No. WO91/15495) have been described as compounds for use as tyrosine kinaseinhibitors for use in the treatment of cancer.

The identification of effective small compounds which specificallyinhibit signal transduction by modulating the activity of receptor andnon-receptor tyrosine kinases to regulate and modulate abnormal orinappropriate cell proliferation is therefore desirable and one objectof this invention.

Finally, certain small compounds are disclosed in U.S. Pat. Nos.5,792,783; 5,834,504; 5,883,113; 5,883,116 and 5,886,020 as useful forthe treatment of diseases related to unregulated TKS transduction. Thesepatents are hereby incorporated by reference in its entirety for thepurpose of disclosing starting materials and methods for the preparationthereof, screens and assays to determine a claimed compound's ability tomodulate, regulate and/or inhibit cell proliferation, indications whichare treatable with said compounds, formulations and routes ofadministration, effective dosages, etc.

As background to the present invention the concept of prodrugs which iswell known in the art. Prodrugs are derivatives of drugs, which afteradministration undergo conversion to the physiologically active species.This conversion may be due caused by hydrolysis in the physiologicalenvironment, or be due to enzymatic hydrolysis. The following literatureis cited: Design of Pro-drugs (Bundgaard H. ed.) 1985 Elsevier SciencePublishers B.V. (Biomedical Devision), Chapter 1; Design ofProdrugs:Bioreversible derivatives for various functional groups andchemical entities (Hans Bundgaard); Bundgaard et al. Int. J. ofPharmaceutics 22 (1984) 45-56 (Elsevier); Bundgaard et al Int. J. ofPharmaceutics 29 (1986) 19-28 (Elsevier); Bundgaard et al. J. Med. Chem.32 (1989) 2503-2507 Chem. Abstracts 95, 138493f (Bungaard et al.); Chem.Abstracts 95, 138592n (Bundgaard et al.); Chem Abstracts 110, 57664p(Alminger et al.) Chem. Abstracts 115, 64029s (Buur et al.); ChemAbstracts 115, 189582y (Hansen et al.); Chem. Abstracts 117, 14347q(Bundgaard et al.); Chem. Abstracts 117, 55790x (Jensen et al.); andChem Abstracts 123, 17593b (Thomsen et al).

BRIEF DESCRIPTION OF THE DRAWING FIGS.

FIG. 1 shows the general scheme for the preparation of the compounds ofthis invention, in particular the compounds of Examples 2-6, 8 and 9.

FIG. 2 shows the general scheme for the preparation of the compounds ofthis invention, in particular the compounds of Examples 12 and 13.

FIG. 3 shows the general scheme for the preparation of the compounds ofthis invention, in particular the compounds of Examples 15, 16, 19 and21.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to organic molecules capable ofmodulating, regulating and/or inhibiting tyrosine kinase signaltransduction. Such compounds are useful for the treatment of diseasesrelated to unregulated TKS transduction, including cell proliferativediseases such as cancer, atherosclerosis, restenosis, metabolic diseasessuch as diabetes, inflammatory diseases such as psoriasis and chronicobstructive pulmonary disease, vascular proliferative disorders such asdiabetic retinopathy, age-related macular degeneration and retinopathyof prematurity, autoimmune diseases and transplant rejection.

DETAILED DESCRIPTION OF THE INVENTION

In one illustrative embodiment, the compounds of the present inventionhave the formula I:

wherein the fragment B represents a tyrosine kinase inhibitor or serinethreonine kinase inhibitor containing a nitrogen atom capable ofreacting with formaldehyde, a substituted aldehyde or substituted ketoneand an amine to provide a compound of formula I and wherein;

R³ and R⁴ are independently selected from the group consisting ofhydrogen, hydrocarbyl and substituted hydrocarbyl radicals, wherein saidsubstituted hydrocarbyl may be substituted with heteroatoms selectedfrom the group consisting of halogen, e.g. fluoro, chloro, bromo, oriodo, nitrogen, phosphorus, sulfur and oxygen, or R³ and R⁴ togetherwith the nitrogen atom may form a cyclic ring, which ring may besubstituted with said heteroatoms, e.g. R³ and R⁴ may be selected fromthe group consisting of hydrogen, alkyl, alkoxy, alkyloxyalkyl, aryl,aryloxy, alkylaryl and alkaryloxy, and

R⁵ and R⁶ are independently selected from the group consisting ofhydrogen, alkyl and aryl radicals. Preferably, R⁵ and R⁶ are hydrogen.

In a preferred embodiment the compounds of the present invention havethe formula II or III:

wherein;

X is O or C(R²)₂;

Y is [C(R²)₂]_(c);

A is NR² or absent;

R¹ is selected from the group consisting of halogen, hydroxy, NO₂, CN,hydrocarbyl and substituted hydrocarbyl radicals, wherein saidsubstituted hydrocarbyl may be substituted with heteroatoms selectedfrom the group consisting of halogen, e.g. fluoro, chloro, bromo, oriodo, nitrogen, phosphorus, sulfur and oxygen, e.g. C₁ to C₄ alkyl andaryl, e.g. phenyl, and when b is 1, R¹ is preferably chloro;

R² is selected from the group consisting of hydrogen, C₁ to C₈ alkyl,(CR⁸R⁹)_(d)C(O)OR¹⁰, COCH₃, CH₂CH₂OH, CH₂CH₂CH₂OH and phenyl;

R is selected from the group consisting of halogen, hydrocarbyl andsubstituted hydrocarbyl radicals, wherein said substituted hydrocarbylmay be substituted with heteroatoms selected from the group consistingof halogen, e.g. fluoro, chloro, bromo, or iodo, nitrogen, phosphorus,sulfur and oxygen, e.g. R may be selected from the group consisting ofhalogen, C₁ to C₈ alkyl, CF₃, OCF₃, OCF₂H, CH₂CN, CN, SR²,(CR⁸R⁹)_(d)C(O)OR², (CR⁸R⁹)_(d)C(O)N(R²)₂, (CR⁸R⁹)_(d)OR², HNC(O)R²,HN—C(O)OR², (CR⁸R⁹)_(d)N(R²)₂, SO₂(CR⁸R⁹)_(d)N(R²)₂, OP(O)(OR²)₂,OC(O)OR², OCH₂O, HN—CH═CH, —N(COR²)CH₂CH₂, HC═N—NH, N═CH—S,O(CR⁸R⁹)_(e)R⁷, (CR⁸R⁹)_(d)R⁷ and —NR₂(CR⁸R⁹)_(e)R⁷ wherein R⁷ isselected from the group consisting of halogen, 3-fluoropyrrolidinyl,3-fluoropiperidinyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl,3-pyrrolinyl, pyrrolidinyl, piperidinyl, methyl isonipecotate,N-(2-methoxyethyl)-N-methylamyl, 1,2,3,6-tetrahydropyridinyl,morpholinyl, hexamethyleneiminyl, piperazinyl-2-one, piperazinyl,N-(2-methoxyethyl)ethylaminyl, thiomorpholinyl, heptamethyleneiminyl,1-piperazinylcarboxaldehyde,2,3,6,7-tetrahydro-(H)-1,4-diazepinyl-5(4H)one, N-methylhomopiperazinyl,(3-dimethylamino)pyrrolidinyl, N-(2-methoxyethyl)-N-propylaminyl,isoindolinyl, nipecotamidinyl, isonipecotamidinyl, 1-acetylpiperazinyl,3-acetamidopyrrolidinyl, trans-decahydroisoquinolinyl,cis-decahydroisoquinolinyl, N-acetylhomopiperazinyl,3-(diethylamino)pyrrolidinyl, 1,4-dioxa-8-azaspiro[4.5]decaninyl,1-(2-methoxyethyl)-piperazinyl, 2-pyrrolidin-3-ylpyridinyl,4-pyrrolidin-3-ylpyridinyl, 3-(methylsulfonyl)pyrrolidinyl,3-picolylmethylaminyl, 2-(2-methylaminoethyl)pyridinyl,1-(2-pyrimidyl)-piperazinyl, 1-(2-pyrazinyl)-piperazinyl,2-methylaminomethyl-1,3-dioxolane,2-(N-methyl-2-aminoethyl)-1,3-dioxolane,3-(N-acetyl-N-methylamino)pyrrolidinyl, 2-methoxyethylaminyl,tetrahydrofurfurylaminyl, 4-aminotetrahydropyran,2-amino-1-methoxybutane, 2-methoxyisopropylaminyl,1-(3-aminopropyl)imidazole, histamyl , N,N-diisopropylethylenediaminyl,1-benzyl-3-aminopyrrolidyl 2-(aminomethyl)-5-methylpyrazinyl,2,2-dimethyl-1,3-dioxolane-4-methanaminyl,(R)-3-amino-1-N-BOC-pyrrolidinyl,4-amino-1,2,2,6,6-pentamethylpiperidinyl, 4-aminomethyltetrahydropyran,ethanolamine and alkyl-substituted derivatives thereof; provided saidalkyl or phenyl radicals may be substituted with one or two halo,hydroxy or lower alkyl amino radicals;

wherein R⁸ and R⁹ may be selected from the group consisting of H,halogen, e.g. F, hydroxy, and C₁-C₄ alkyl or CR⁸R⁹ may represent acarbocyclic ring of from 3 to 6 carbons, preferably R⁸ and R⁹ are H orCH₃, preferably in the compounds of Formula III when a is 1, R isdimethyl amino and in the compounds of Formula II when a is 1, R ismorpholonyl;

R³, R⁴, R⁵ and R⁶, are as defined above;

R¹⁰ is hydrogen, C₁ to C₈ alkyl or arylalkyl;

a is 0 or an integer of from 1 to 3;

b is 0 or an integer of from 1 to 3;

c is an integer of from 1 to 2;

d is 0 or an integer of from 1 to 5

e is an integer of from 2 to 5

the wavy line represents a E or Z bond; and

Ar is selected from the group consisting of aryl, substituted aryl,heteroaryl, and substituted heteroaryl, wherein said substitutedhydrocarbyl or said substituted heteroaryl may be substituted withheteroatoms selected from the group consisting of halogen, e.g. fluoro,chloro, bromo, or iodo, nitrogen, phosphorus, sulfur and oxygen, e.g. Armay be selected from the group consisting of monocyclic and bicyclicaryl and heteroaryl, including both fused and non-fused dicyclic aryl orheteroaryl, e.g. phenyl, naphthyl, pyridyl, pyrrolyl, luryl, thienyl,etc. and substituted derivatives thereof; and pharmaceuticallyacceptable salts thereof. Preferably, Ar is a monocyclic aryl orheteroaryl, e.g., phenyl or pyrrolyl.

Preferably R⁵ and R⁶ are hydrogen.

Preferably R³ is H and R⁴ is selected from the group consisting ofalkyl, e.g. n-butyl, or alkyloxyalkyl, e.g. methyloxypropyl, or R³ andR⁴, together with the nitrogen atom forms a cyclic ring having 5 or 6members, e.g., a 6 member ring, which may include an enchained oxygen ornitrogen atom, e.g. R³ and R⁴ may, together with the nitrogen atom maybe morpholinyl or piperidinyl and said morpholinyl or said piperidinylring may be substituted with one or more lower alkyl groups, e.g.,methyl.

Preferably, X is O and Y is CH₂ and R may be di(lower)alkyl amino, e.g.,dimethyl amino.

Preferably, Ar is phenyl or pyrrolyl and R may be lower alkyl, e.g.,methyl, or morpholinyl.

In one preferred embodiment of the invention, the R³ and R⁴ togetherwith the nitrogen atom form a cyclic ring having from 3 to 8, e.g., 5 or6, members and more preferably said cyclic ring includes an enchainedoxygen atom or a second nitrogen atom. That is, R³ and R⁴ together withthe nitrogen atom may be pyrrolidinyl, piperidinyl, piperazinyl ormorpholinyl.

In another preferred embodiment R³ is hydrogen and R⁴ is alkyl oralkyloxyalkyl.

In one preferred embodiment of formula II A is —NH—, Ar is

wherein R₃′, and R₄′ are each independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl,alkaryloxy, halogen, trihalomethyl, S(O)R², SO₂(R²)₂, SO₃R², SR², NO₂,N(R²)₂, OH, CN, C(O)R², OC(O)R², NHC(O)R², (CH₂)_(d)CO₂R², and(CH₂)_(d)CON(R²)₂;

R′ is hydrogen, alkyl, aryl, alkylaryl, haloalkyl, (CR⁸R⁹)_(d)C(O)OR²,(CR⁸R⁹)OR², or (CR⁸R⁹)_(e)N(R²)₂, or (CR⁸R⁹)_(e)R⁷, wherein d, e, R²,R⁷, R⁸ and R⁹ are as defined above.

In another preferred embodiment of formula II A is —NH—, Ar is

wherein R₃′ and R₄′, are as defined above, R¹¹ is R¹ or R¹¹ takentogether with the nitrogen atom may be a 5 or 6 membered ring which mayhave an enchained oxygen atom or a second nitrogen atom, for examplemorpholinyl, piperidinyl piperzinyl, etc.

In yet another preferred embodiment of formula II, A is absent, Ar is afive membered heteroaryl ring selected from the group consisting offuryl, thiophene, pyrrole, 2,4-dimethylpynole,2,4-dimethyl-3-pyrrole-propionic acid, pyrazole, imidazole,1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole,isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole,1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole,1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiatriazole, and tetrazole, optionallysubstituted at one or more positions with R², O(CR⁸R⁹)_(e)N(R²)₂,(CR⁸R⁹)_(d)N(R²)₂ or NR²(CR⁸R⁹)_(e)N(R²)₂ (CR⁸R⁹)_(d)C(O)OR²,O(CR⁸R⁹)_(e)R⁷, (CR⁸R⁹)_(d)R⁷ and NR²(CR⁸R⁹)_(e)R⁷, wherein d, e, R²,R⁷, R⁸ and R⁹ are as defined above.

In yet another preferred embodiment of formula II A is —NH—, Ar is afive membered heteroaryl ring selected from the group consisting offuryl, thiophene, pyrrole, 2,4-dimethylpyrrole, pyrazole, imidazole,1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole,isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole,1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole,1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiatriazole, and tetrazole, optionallysubstituted at one or more positions with with R², O(CR⁸R⁹)_(e)N(R²)₂,(CR⁸R⁹)_(d)N(R²)₂, NR²(CR⁸R⁹)_(e)N(R²)₂, (CR⁸R⁹)_(d)C(O)OR²,O(CR⁸R⁹)_(e)R⁷, (CR⁸R⁹)_(d)R⁷ and NR²(CR⁸R⁹)_(e)R⁷ and wherein d, e, R²,R⁷, R⁸ and R⁹ are as defined above.

In a preferred embodiment of formula ER; X is O or CH₂;

Y is [C(R²)₂]_(c);

R¹ is selected from the group consisting of halogen, hydroxy, C₁ to C₄alkyl;

R² is selected from the group consisting of hydrogen, C₁ to C₈ alkyl,(CR⁸R⁹)_(d)C(O)OR¹⁰;

R is selected from the group consisting of halogen, C₁ to C₈ alkyl, CF₃,OCF₃, OCF₂H, (CR⁸R⁹)_(d)C(O)OR², (CR⁸R⁹)_(d)C(O)N(R²)₂, HNC(O)R²,HN—C(O)OR², (CR⁸R⁹)_(d)N(R²)₂, SO₂(CR⁸R⁹)_(d)N(R²⁾ ₂, O(CR⁸R⁹)_(e)R⁷ and(CR⁸R⁹)_(d)R⁷, —NR²(CR⁸R⁹)_(e)R⁷ wherein d, e, R², R⁷, R⁸ and R⁹ are asdefined above.

The present invention is further directed to pharmaceutical compositionscomprising a pharmaceutically effective amount of the above-describedcompounds and a pharmaceutically acceptable carrier or excipient. Such acomposition is believed to modulate signal transduction by a tyrosinekinase, either by inhibition of catalytic activity, affinity to ATP orability to interact with a substrate.

More particularly, the compositions of the present invention may beincluded in methods for treating diseases comprising proliferation,fibrotic or metabolic disorders, for example cancer, fibrosis,psoriasis, atherosclerosis, arthritis, and other disorders related toabnormal vasculogenesis and/or angiogenesis, such as diabeticretinopathy.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the free bases and whichare obtained by reaction with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like.

“Alkyl” refers to a straight-chain, branched or cyclic saturatedaliphatic hydrocarbon. Preferably, the alkyl group has 1 to 12 carbons.More preferably, it is a lower alkyl of from 1 to 7 carbons, mostpreferably 1 to 4 carbons. Typical alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl andthe like. The alkyl group may be optionally substituted with one or moresubstituents are selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino, and SH.

“Alkenyl” refers to a straight-chain, branched or cyclic unsaturatedhydrocarbon group containing at least one carbon-carbon double bond.Preferably, the alkenyl group has 1 to 12 carbons. More preferably it isa lower alkenyl of from 1 to 7 carbons, most preferably 1 to 4 carbons.The alkenyl group may be optionally substituted with one or moresubstituents selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino, and SH.

“Alkynyl” refers to a straight-chain, branched or cyclic unsaturatedhydrocarbon containing at least one carbon-carbon triple bond.Preferably, the alkynyl group has 1 to 12 carbons. More preferably it isa lower alkynyl of from 1 to 7 carbons, most preferably 1 to 4 carbons.The alkynyl group may be optionally substituted with one or moresubstituents selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino, and SH.

“Alkoxyl” refers to an “O-alkyl” group.

“Aryl” refers to an aromatic group which has at least one ring having aconjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups. The aryl group may be optionallysubstituted with one or more substituents selected from the groupconsisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO₂, amine,thioether, cyano, alkoxy, alkyl, and amino.

“Alkaryl” refers to an alkyl that is covalently joined to an aryl group.Preferably, the alkyl is a lower alkyl.

“Carbocyclic aryl” refers to an aryl group wherein the ring atoms arecarbon.

“Heterocyclic aryl” refers to an aryl group having from 1 to 3heteroatoms as ring atoms, the remainder of the ring atoms being carbon.Heteroatoms include oxygen, sulfur, and nitrogen. Thus, heterocyclicaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkylpyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.

“Hydrocarbyl” refers to a hydrocarbon radical having only carbon andhydrogen atoms. Preferably, the hydrocarbyl radical has from 1 to 20carbon atoms, more preferably from 1 to 12 carbon atoms and mostpreferably from 1 to 7 carbon atoms.

“Substituted hydrocarbyl” refers to a hydrocarbyl radical wherein one ormore, but not all, of the hydrogen and/or the carbon atoms are replacedby a halogen, nitrogen, oxygen, sulfur or phosphorus atom or a radicalincluding a halogen, nitrogen, oxygen, sulfur or phosphorus atom, e.g.fluoro, chloro, cyano, nitro, hydroxyl, oxa, oxo, phosphate, thiol, etc.

“Amide” refers to —C(O)—NH—R, wherein R is alkyl, aryl, alkylaryl orhydrogen.

“Thioamide” refers to —C(S)—NH—R, wherein R is alkyl, aryl, alkylaryl orhydrogen.

“Amine” refers to a —N(R′)R″ group, wherein R′ and R″ are independentlyselected from the group consisting of alkyl, aryl, and alkylaryl.

“Thioether” refers to —S—R, wherein R is alkyl, aryl, or alkylaryl.

“Sulfonyl” refers to —S(O)₂—R, where R is aryl, C(CN)═C-aryl, CH₂CN,alkyaryl, sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl.

In particular the compounds of the present invention are selected fromthe compounds of Table 1-Table 3, below.

TABLE 1 Substituted1-Aminomethyl-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro- indol-2-oneAnalogs

Example # R¹ NR₃R₄ R″ R′″ 2 H N(CH₂)₅ H H 3 H N(CH₂)₂O(CH₂)₂ H H 4 HN(CH₂)₂N(CH₃)(CH₂)₂ H H 5 H NHCH₂CH₂CH₂OCH₃ H H 6 H NHCH₂CH₂CH₂CH₃ H H 85-Cl N(CH₂)₅ CH₃ CH₃ 9 5-Cl N(CH₂)₂O(CH₂)₂ CH₃ CH₃

Table 2: Substituted1-Aminomethyl-3-[(4-morpholin-4-yl-phenylamino)-methylene]-1,3-dihydro-indol-2-oneAnalogs

TABLE 2 Substituted 1-Aminomethyl-3-[(4-morpholin-4-yl-phenylamino)-methylene]-1,3-dihydro-indol-2-one Analogs

Example # NR₃R₄ 12 N(CH₂)₅ 13 N(CH₂)₂O(CH₂)₂

Table 3: Substituted1-Aminomethyl-3-(3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-oneAnalogs

TABLE 3 Substituted1-Aminomethyl-3-(3H-isobenzofuran-1-ylidene)-1,3-dihydro- indol-2-oneAnalogs

Example # R_(b) ¹ Ra NR₃R₄ 15 H H N(CH₂)₅ 16 H H N(CH₂)₂O(CH₂)₂ 19 HN(CH₃)₂ N(CH₂)₅ 21 5-Cl N(CH₃)₂ N(CH₂)₅

Note in Examples 15, 16 and 19, b is 0, therefore R¹ is given as H. InExamples 15 and 16 a is 0, therefore R is given as H.

The present invention relates to compounds capable of regulating and/ormodulating tyrosine kinase signal transduction and more particularlyreceptor and non-receptor tyrosine kinase signal transduction.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and phosphorylation. Bindingsites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects and responses to theextracellular microenvironment).

It has been shown that tyrosine phosphorylation sites in growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Several intracellular substrate proteinsthat associate with receptor tyrosine kinases have been identified. Theymay be divided into two principal groups: (1) substrates which have acatalytic domain; and (2) substrates which lack such domain but serve asadapters and associate with catalytically active molecules. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. These observations suggest that the functionof each receptor tyrosine kinase is determined not only by its patternof expression and ligand availability but also by the array ofdownstream signal transduction pathways that are activated by aparticular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

Tyrosine kinase signal transduction results in, among other responses,cell proliferation, differentiation and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis,arthritis and diabetic retinopathy (or other disorders related touncontrolled angiogenesis and/or vasculogenesis, e.g. maculardegeneration).

This invention is therefore directed to compounds which regulate,modulate and/or inhibit tyrosine kinase signal transduction by affectingthe enzymatic activity of the RTKs and/or the non-receptor tyrosinekinases and interfering with the signal transduced by such proteins.More particularly, the present invention is directed to compounds whichregulate, modulate and/or inhibit the RTK and/or non-receptor tyrosinekinase mediated signal transduction pathways as a therapeutic approachto cure many kinds of solid tumors, including but not limited tocarcinoma, sarcoma, leukemia, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastona. Indications may include, but arenot limited to brain cancers, bladder cancers, ovarian cancers, gastriccancers, pancreas cancers, colon cancers, blood cancers, lung cancersand bone cancers.

Chemical Stability

The chemical stability of Example 12 of the invention has been studiedusing buffers at pH 1, pH 3 and pH 7. Example 12 prepared inacetonitrile at 200 μg/mL and diluted to 20 μg/mL in buffers at pH 1, pH3 and pH 7. Example 12 was measured to have a half—life (t_(1/2)) ofapproximately 20 hours at pH 3 and 38 hours at pH 1. The half—life(t_(1/2)) of Example 12 at pH 7.4 was less than 30 minutes.

Biological data for the compounds of the present invention was generatedby use of the following assays.

VEGF Stimulated Ca⁺⁺ Signal in Vitro

Automated FLIPR (Fluorometric Imaging Plate Reader) technology was usedto screen for inhibitors of VEGF induced increases in intracellularcalcium levels in fluorescent dye loaded endothelial cells. HUVEC (humanumbilical vein endothelial cells) (Clonetics) were seeded in 96-wellfibronectin coated black-walled plates overnight at 37° C./5% CO₂. Cellswere loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cellswere washed 4 times (Original Cell Wash, Labsystems) to removeextracellular dye. Test compounds were reconstituted in 100% DMSO andadded to the cells to give a final DMSO concentration of 0.1%. Forscreening, cells were pre-incubated with test agents for 30 minutes, ata single concentration (10 μM) or at concentrations ranging from 0.01 to10.0 μM followed by VEGF stimulation (5 ng/mL). Changes in fluorescenceat 516 nm were measured simultaneously in all 96 wells using a cooledCCD camera. Data were generated by determining max-min fluorescencelevels for unstimulated, stimulated, and drug treated samples. IC₅₀values for test compounds were calculated from % inhibition of VEGFstimulated responses in the absence of inhibitor.

VEGFR2 Kinase Assay

The cytoplasmic domain of the human VEGF receptor (VEGFR-2) wasexpressed as a Histidine-tagged fusion protein following infection ofinsect cells using an is engineered baculovirus. His-VEGFR-2 waspurified to homogeneity, as determined by SDS-PAGE, using nickel resinchromatography. Kinase assays were performed in 96 well microtiterplates that were coated overnight with 30 μg of poly-Glu-Tyr (4:1) in 10mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates wereincubated with 1% BSA and then washed four times with PBS prior tostarting the reaction. Reactions were carried out in 120 μL reactionvolumes containing 3.6 μM ATP in kinase buffer (50 mM Hepes buffer pH7.4, 20 mM MgCl₂, 0.1 mM MnCl₂ and 0.2 mM Na₃VO₄). Test compounds werereconstituted in 100% DMSO and added to the reaction to give a finalDMSO concentration of 5%. Reactions were initiated by the addition 0.5ng of purified protein. Following a ten minute incubation at 25° C., thereactions were washed four times with PBS containing 0.05% Tween-20. 100μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugatewas diluted 1:10000 in PBS-Tween-20 and added to the wells for 30minutes. Following four washes with PBS-Tween-20, 100 μl ofO-phenylenediamine Dihydrochloride in Phosphate-citrate buffer,containing urea hydrogen peroxide, was added to the wells for 7 minutesas a colorimetric substrate for the peroxidase. The reaction wasterminated by the addition of 100 μl of 2.5N H₂SO₄ to each well and readusing a microplate ELISA reader set at 492 nm. IC₅₀ values for compoundinhibition were calculated directly from graphs of optical density(arbitrary units) versus compound concentration following subtraction ofblank values.

VEGF-induced Dermal Extravasation in Guinea Pig (Miles Assay).

Male Hartley guinea pigs (300-600 g) were anesthetized with isofluorane,sheared, and given a single dose of drug or the respective vehicle. Theguinea pigs were dosed orally unless indicated otherwise in Table 3. Tenminutes prior to the end of drug treatment, guinea pigs wereanesthetized with isofluorane, and 0.5% Evans blue dye (EBD) in PBS(13-15 mg/kg dose of EBD) was injected intravenously. After 5 minutes,triplicate intradermal injections of 100 ng rhVEGF₁₆₅ in 100 μl PBS andof 100 μl PBS alone were administered on the flank. After 20 minutes,each animal was cuthanized with Pentosol, and the skin containing theintradermal injection sites was removed for image analysis.

Using an analog video camera coupled to a PC, an image of eachtrans-illuminated skin sample was captured, and the integrated opticaldensity of each injection site was measured using ImagePro 4. For eachskin sample, the difference between the mean optical density of the VEGFsites and mean optical density of the PBS sites is the measure ofVEGF-induced EBD extravasation in that animal. These measured valueswere averaged per study group to determine the mean VEGF-induced EBDextravasation for each experimental condition, and the group means werethen compared to assess inhibition of VEGF-induced EBD extravasation inthe drug-treated groups relative to the vehicle-treated controls.

To determine the dose required for 50% inhibition (ID₅₀), the percentinhibition data was plotted as a function of oral dose, using the‘best-fit’ analysis within MicroSoft Excel software. The ID₅₀ value wasverified visually by using the plotted data (horizontal line from 50% yvalue, at intersection with best-fit line drop vertical line to x axis(dose).

Laser-induced Choroidal Neovascularization (CNV) in Rat (CNV Assay).

CNV was induced and quantified in this model as previously described(Edelman and Castro. Exp. Eye Res. 2000; 71:523-533). On day 0, maleBrown Norway rats (200-300 g) were anesthetized with 100 mg/kg Ketamineand 10 mg/kg Xylazine, and pupils were dilated with 1% Tropicamide.Using the blue-green setting of a Coherent Novus Argon Laser, 3 laserbums (90 mW for 0.1 s; 100 μm diameter) were given to each eye betweenthe retinal vessels around the optic nerve head. Rats were dosed withtest compounds in their indicated vehicles orally once daily.

On day 10, rats were sacrificed with 100% CO₂, and blood vessels werelabeled by vascular perfusion with 10 mg/ml FITC-dextran (MW 2×10⁶).Using an epifluorescence microscope (20×) coupled to a spot digitalcamera and a PC, images were obtained from the flat mounts of theRPE-choroid-sclera from each eye, and the area occupied byhyperfluorescent neovessels within each laser lesion was measured usingImagePro 4 software.

To determine the dose required for 50% inhibition (ID₅₀), the percentinhibition data was plotted as a function of oral dose, using the‘best-fit’ analysis within MicroSoft Excel software. The ID₅₀ value wasverified visually by using the plotted data (horizontal line from 50% yvalue, at intersection with best-fit line drop vertical line to x axis(dose).

The results of said assays are set forth in Table 4-6, below.

TABLE 4 VEGF Stimulated Ca⁺⁺ Signal Assay and VEGF Kinase Assay DataVEGF Stimulated Ca⁺⁺ VEGFR2 Kinase Assay Example # Signal Assay IC₅₀(nM) IC₅₀ (nM) 11 294 260 12 432 478 13 722 3858 14 110 15 16 18 22 3119 120 133 20 33 22

TABLE 5 VEGF-induced Dermal Extravasation in Guinea Pig (Miles Assay)Results Example # Vehicle % Inhibition @ Dose (mg/Kg) ID₅₀ 11 corn oil40% @ 75 mg/Kg 12 corn oil 88% @ 75 mg/Kg 36 mg/Kg 13 corn oil 35% @ 75mg/Kg

TABLE 6 Rat Laser Choroidal Angiogenesis Assay Results Example # Vehicle% Inhibition @ Dose (mg/Kg) ID₅₀ 12 corn oil 94% @ 80 mg/Kg (sid) 36mg/Kg sid = once daily dosing

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Preparation ofZ-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one

A mixture of oxindole (137 mg, 1.03 mmol), pyrrole-2-carboxaldehyde(115.1 mg, 1.21 mmol) and piperidine (40 μL, 0.404 mmol) in 2.0 mL MeOHwas heated at reflux for 3 h. The mixture was cooled in an ice bath andthe solid which formed was collected by filtration to give the titlecompounds (208 mg, 96%) as a yellow solid.

EXAMPLE 2 Preparation of1-Piperidin-1-ylmethyl-3-(1-H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one

A solution of Z-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (0.60g, 2.85 mmol), paraformaldehyde (0.13 g, 4.28 mmol) and piperidine (0.24g, 2.85 mmol) in EtOH (5 mL) was heated to 60-70° C. for 14 h. Thereaction mixture was cooled to −20° C. for 1 h and the fine yellow solidpresent was collected by filtration. The solid collected was dried undervacuum to give the title compound as a yellow solid (0.68 g, 77%).

¹H NMR (500 MHz, d₆-DMSO) δ 13.17 (br s, 1H), 7.81 (s, 1H), 7.67 (d,J=7.3 Hz, 1H), 7.37 (m, 1H), 7.22 (m, 1H), 7.18 (d, J=7.3 Hz, 11), 7.06(ddd, J=7.3, 7.3, 1.2 Hz, 1H), 6.88 (m, 1H), 6.37 (m, 1H), 4.58 (s, 2H),2.54 (br s, 4H), 1.46 (m, 4H), 1.33 (br m, 2H).

EXAMPLE 3 Preparation of1-Morpholin-4-ylmethyl-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one

A mixture of Z-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (120mg, 0.57 mmol), paraformaldehyde (32 mg, 1.1 mmol), and morpholine (62.2μL, 0.71 mmol) in 8.0 mL of 20% dioxane in EtOH was heated at reflux for19.5 h. The reaction mixture was concentrated and paraformaldehyde (10.0mg, 0.33 mmol), morpholine (15 μL, 0.17 mmol) and EtOH (7 mL) was added.The reaction mixture refluxed for 24 h. The reaction mixture was cooledto room temperature and concentrated. The residue was dissolved in 15 mLof CHCl₃ and treated with activated charcoal. The resulting suspensionwas filtered and the filtrate concentrated. The residue wascrystallization from ethyl acetate/hexane to give the title compound(88.0 mg, 50%) as a bright yellow solid.

¹H NMR (500 MHz, d₆-DMSO) δ 13.03 (br s, 1H), 7.80 (s, 1H), 7.66 (d,J=7.3 Hz, 1H), 7.35 (br s, 1H), 7.19 (m, 2H), 7.05 (m, 1H), 6.86 (m,1H), 6.35 (m, 1H), 4.56 (s, 2H), 3.52 (m, 4H), 2.53 (m, 4H).

EXAMPLE 4 Preparation of1-(4-Methyl-piperazin-1-ylmethyl)-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one

A mixture of Z-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (120mg, 0.57 mmol), paraformaldehyde (17 mg, 0.57 mmol), and1-methylpiperazine (63 μL, 0.57 mmol) in 4.0 mL EtOH was heated atreflux for 6.5 h. After standing at room temperature for 23 h, themixture was concentrated to 3 mL solvent and 1 mL of hexane added. Theresulting solution was cooled to 0° C. and the yellow solid which formedwas collected by filtration. The solid collected was partitioned between30 mL dilute HCl and 20 mL Ethyl acetate. The organic layer was removedand the aqueous layer washed with ethyl acetate (15 mL). The aqueouslayer was made basic to pH 8 with saturated NaHCO₃ and extracted withethyl acetate. The ethyl acetate layer was washed with H₂O, brine anddried with Na₂SO₄. The organic layer was concentrated and the residuewas crystallized from ethyl acetate/hexane to give the title compound(38 mg, 21%) as a bright yellow solid.

¹H NMR (500 MHz, CDCl₃) δ 13.35 (br s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.42(s, 1H), 7.19 (dd, J=7.8, 7.6 Hz, 1H), 7.14 (br s, 1H), 7.06 (dd, J=7.6,7.6 Hz, 1H), 7.02 (d, J=7.8 Hz, 1H), 6.75 (m, 1H), 6.36 (m, 1H), 4.57(s, 2H), 2.69 (br s, 4H), 2.41 (br s, 4H), 2.24 (s, 3H).

EXAMPLE 5 Preparation of1-[(3-Methoxy-propylamino)-methyl]-3-(1H-pyrrol-2-ylmethylene-1,3-dihydro-indol-2-one

A mixture of Z-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (120.0mg, 0.571 mmol), paraformaldehyde (17.1 mg, 0.571 mmol), and3-methoxypropylamine (58.2 μL, 0.571 mmol) in 4.0 mL EtOH was heated atreflux for 6.5 h. The reaction mixture was cooled to room temperatureand let stand for 22 h. The reaction mixture was filtered and the filtercake rinsed with 30% ethyl acetate in hexane. The filtrate wasconcentrated in vacuo and 4 mL of EtOH added. The reaction was refluxedan additional 19.5 h. The reaction mixture was cooled to roomtemperature and partitioned between 30 mL dilute HCl and 20 mL Ethylacetate. The organic layer was removed and the aqueous layer washed with15 mL ethyl acetate. The aqueous layer was made basic to pH 8 withsaturated NaHCO₃ and extracted with ethyl acetate. The ethyl acetatelayer was washed with H₂O, brine and dried over anhydrous Na₂SO₄. Theethyl acetate layer was filtered and then concentrated. The solidobtained (42.5 mg) was dissolved in CHCl₃ and purified by chromatography(silica gel, 1:1/hexane:acetone) to give the title compound (1.5 mg,1%).

¹H NMR (500 MHz, CDCl₃) δ 13.40 (br s, 1H), 7.50 (d, J=7.5 Hz, 1H), 7.44(s, 1H), 7.21 (m, 1H), 7.16 (m, 1H), 7.07 (m, 1H), 6.95 (d, J=7.7 Hz,1H), 6.77 (m, 1H), 6.38 (m, 1H), 4.81 (s, 2H), 3.37 (t, J=6.3 Hz, 2H),3.24 (s, 3H), 2.69 (t, J=6.7 Hz, 2H), 1.72 (quintet, J=6.5 Hz, 2H).

EXAMPLE 6 Preparation of1-Butylaminomethyl-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one

A mixture of Z-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (120.0mg, 0.571 mmol), paraformaldehyde (17.1 mg, 0.571 mmol), and butylamine(56.4 μL, 0.571 mmol) in 5.5 mL of EtOH was heated at reflux for 2.5 h.Then reaction mixture was treated with 2 mL of hexane was and thencooled to 0° C. The reaction mixture was then filtered to removeZ-(1H-Pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one. The filtrate waspartitioned between dilute HCl and ethyl acetate. The organic layer wasremoved and the aqueous layer washed with 15 mL Ethyl acetate. Theaqueous phase was made basic to pH 8 with saturated NaHCO₃ and extractedwith ethyl acetate. The ethyl acetate layer was washed with H₂O, brineand dried over anhydrous Na₂SO₄. The solvent was removed in vacuo togive 58 mg of yellow oil. The oil was purified by chromatography (silicagel, 1:1/hexane:ethyl acetate) to give the title compound (51.6 mg, 31%)as a yellow solid.

¹H NMR (500 MHz, CDCl₃) δ 13.41 (br s, 1H), 7.51 (d, J=7.5 Hz, 1H), 7.46(s, 1H), 7.23 (m, 1H), 7.17 (br s, 1H), 7.09 (m, 1H), 6.96 (d, J=7.7 Hz,1H), 6.78 (m, 1H), 6.39 (m, 1H), 4.83 (s, 2H), 2.61 (t, J=7.1 Hz, 2H),1.43 (m, 2H), 1.29 (m, 2H), 0.86 (t, J=7.3 Hz, 3H).

EXAMPLE 7 Preparation of5-chloro-3-(1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one

A mixture of 5-chlorooxindole (6.98 g, 41.6 mmol),3,5-dimethyl-1H-pyrrole-2-carhoxaldehyde (5.12 g, 41.6 mmol) andpiperidine (410 μL, 4.16 mmol) in 200 mL of EtOH was heated at refluxfor 8 h. The reaction mixture was cooled to room temperature andfiltered to give the title compound (4.50 g, 40%) as a red/orange solid.

EXAMPLE 8 Preparation of5-Chloro-3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-one

A suspension of5-chloro-3-(1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one (260 mg,0.96 mmol), piperidine (95 μL, 0.96 mmol) and paraformaldehyde (43 mg,1.44 mmol) in 5 mL of dioxane and 5 mL of EtOH was heated to 80° C. for15 h. The resulting solution was cooled to room temperature followed bycooling to −20° C. for 1 h. The solid which formed was collected byfiltration and rinsed with ETOH (previously cooled to −20° C). The solidcollected was dried under vacuum to give the title compound (180 mg,51%) as a yellow-orange solid.

EXAMPLE 9 Preparation of5-Chloro-3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1-morpholin-4-ylmethyl-1,3-dihydro-indol-2-one

A suspension of5-chloro-3-(1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one (120 mg,0.44 mmol), morpholine (39 μL, 0.44 mmol) and paraformaldehyde (20 mg,0.66 mmol) in 8 mL of dioxane and 2 mL of EtOH was heated to 80° C. for15 h. The reaction mixture was cooled to room temperature and treatedwith morpholine (25 μL, 0.28 mmol) and paraformaldehyde (16 mg, 0.50mmol) The reaction mixture was heated at 80° C. for 4 h. The reactionmixture was cooled to room temperature and partially concentrated undera nitrogen stream. The reaction mixture was cooled to −20° C. The solidwhich formed was collected by filtration and rinsed with EtOH(previously cooled to −20° C.). The solid collected was dried undervacuum to give the title compound (85 mg, 52%) as an orange solid.

EXAMPLE 10 Preparation of 3-Hydroxymethylene-1,3-dihydro-indol-2-one

To a slurry of oxindole (1.33 g, 10.0 mmol) and ethyl formate (2.42 mL,30.0 mmol) was added 4.85 mL of 21 wt. % NaOEt in EtOH. The thicksolution was stirred for 30 min at room temperature and then heated atreflux for 30 min. The solution was acidified to pH=3 with 10% aqueousHCl and 5 mL H₂O was added. The solid which formed was filtered andrinsed with H₂O to give the title compound (1.34 g, 83%) as a paleyellow solid.

EXAMPLE 11 Preparation of3-[(4-morpholinophenylamino)-methylene]-1,3-dihydro-indol-2-one

A solution of 4-morpholinoaniline (8.3 g, 51.5 mmol) in 100 mL oftetrahydrofuran was treated with3-hydroxymethylene-1,3-dihydro-indol-2-one (5.5 g, 30.9 mmol) in oneportion. The reaction mixture was heated at reflux for 3 h. The reactionmixture was concentrated. The residue was treated with 125 mL of ethylacetate and the resulting suspension was heated at 40° C. for 2 h. Thesuspension was cooled to room temperature and the solid collected bysuction filtration and washed with ethyl acetate. The solid obtained wasdried under vacuum to give the title compound (10.1 g, 92%) as a yellowsolid.

EXAMPLE 12 Preparation of3-[(4-Morpholin-4-yl-phenylamino)-methylene]-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-one

A solution of3-[(4-morpholinophenylamino)-methylene]-1,3-dihydro-indol-2-one (17.3 g,53.8 mmol) and paraformaldehyde (2.43 g, 80.9 mmol.) in 125 mL of EtOHwas treated with piperidine (5.87 mL, 59.3 mmol). The reaction mixturewas then heated at a reflux temperature for 5 hours during which time ayellow precipitate formed. The reaction mixture was allowed to cool toroom temperature and the solid was collected by filtration and washedwith EtOH and dried under vacuum to give the title compound (21.5 g,95%) as a yellow solid.

EXAMPLE 13 Preparation of1-Morpholin-4-ylmethyl-3-[(4-morpholin-4-yl-phenylamino)-methylene]-1,3-dihydro-indol-2-one

A solution of3-[(4-morpholinophenylamino)-methylene]-1,3-dihydro-indol-2-one (0.71 g,2.21 mmol) and paraformaldehyde (0.10 g, 3.33 mmol.) in 8 mL of EtOH wastreated with morpholine (213 μL, 2.44 mmol). The reaction mixture wasthen heated at a reflux temperature overnight during which time a yellowprecipitate formed. The reaction mixture was allowed to cool to roomtemperature and the solid was collected by filtration and washed withEtOH and dried under vacuum to give the title compound (0.769 g, 83%) asa yellow solid.

EXAMPLE 14 Preparation of3-(3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-one

To a suspension of sodium hydride (6.0 g, 150 mmol, 60% in mineral oil)in 300 mL DMF was added oxindole (10.0 g, 75.1 mmol) in 50 mL DMF over 8min. After stirring for 15 min at room temperature, a solution ofphthalide (13.1 g, 97.6 mmol) in 50 mL DMF was added over 1 min. Themixture was stirred for 1.25 h, then poured into 1100 mL H₂O. Additionof 4% aqueous HCl solution gave a yellow solid which was filtered andrinsed with H₂O to give the title compound (8.75 g, 47%).

¹H NMR (500 MHz, d₆-DMSO) δ 10.41 (s, 1H), 9.65 (d, J=8.1 Hz, 1H), 7.83(d, J=7.6 Hz, 1H), 7.65 (m, 2H), 7.55 (m, 1H), 7.10 (ddd, J=7.6, 7.6,1.0 Hz, 1H), 6.95 (ddd, J=7.6, 7.6, 1.0 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H),5.81 (s, 2H).

EXAMPLE 15 Preparation of3-(3H-Isobenzofuran-1-ylidene)-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-one

A mixture of 3-(3H-isobenzofuran-1-ylidene)1,3-dihydro-indol-2-one (4.32g, 17.3 mmol), paraformaldehyde (0.99 g, 32.9 mmol), and piperidine(2.14 mL, 21.7 mmol) in 192 mL EtOH and 48 mL dioxane was heated atreflux for 18.5 h. The solution was concentrated in vacuo to a volume of100 mL and then refluxed for 1 h to dissolve the precipitant. Themixture was allowed to cool to room temperature and the precipitatefiltered to give the title compound (3.728 g) as a bright yellow solid.An additional 0.52 g of the title compound was obtained bycrystallization of the filtrate from ethyl acetate. The two lots werecombined to give 4.248 g (71%) of the title compound as a bright yellowsolid.

¹H NMR (500 MHz, d₆-DMSO) δ 9.67 (d, J=8.1 Hz, 1H), 7.90 (d, J=7.8 Hz,1H), 7.68 (m, 2H), 7.58 (m, 1H), 7.18 (dd, J=7.8, 7.6 Hz, 1H), 7.12 (d,J=7.6 Hz, 1H), 7.03 (dd, J=7.6, 7.6 Hz, 1H), 5.83 (s, 2H), 4.50 (s, 2H),2.52 (br s, 4H), 1.45 (m, 4H), 1.32 (br s, 2H).

EXAMPLE 16 Preparation of3-(3H-Isobenzofuran-1-ylidene)-1-morpholin-4-ylmethyl-1,3-dihydro-indol-2-one

A mixture of 3-(3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-one(4.32 g, 17.3 mmol), paraformaldehyde (0.99 g, 32.9 mmol), andmorpholine (1.89 mL, 21.7 mmol) in 192 mL EtOH and 48 mL dioxane washeated at reflux for 18.5 h. The solution was concentrated in vacuo to avolume of 100 mL and then refluxed for 1 h to dissolve the precipitate.The mixture was allowed to cool to room temperature and the solid whichformed was collected by filtration to give 4.19 g of a bright yellowsolid. An additional 0.72 g of yellow solid was obtained bycrystallization of the filtrate from ethyl acetate. The combinedmaterial was crystallized from ethyl acetate give the title compound(2.49 g, 41%) as a fine yellow needles.

¹H NMR (500 MHz, d₆-DMSO) δ 9.67 (d, J=7.8 Hz, 1H), 7.91 (d, J=7.6Hz,1H), 7.68 (m, 2H), 7.58 (m, 1H), 7.19 (m, 1H), 7.14 (d, J=7.3 Hz, 1H),7.05 (m, 1H), 5.84 (s, 2H), 4.53 (s, 2H), 3.53 (m, 4H), 2.54 (br s, 4H).

EXAMPLE 17 Preparation of 5-Dimethylaminophthalide

Sodium cyanoborohydride (8.42 g, 134 mmol) was added into a stirredsuspension of 5-aminophthalide (5.0 g, 33.5 mmol) and 37% CH₂O/H₂Osolution (24.9 mL, 335 mmol) in 120 mL of acetonitrile. The mixture wasstirred at room temperature for 1 hour, cooled to 0 C and 120 mL of 10%aqueous acetic acid solution was added. The mixture was then stirred atroom temperature for 1 hour, evaporated under low pressure until noacetonitrile remained. The resulting mixture was extracted with ethylacetate (2×125 mL). The combined organic extracts were washed withsaturated NaHCO₃ solution (125 mL) and brine (125 mL), dried over Na₂SO₄and evaporated to dryness to give a light brown solid, which wastriturated with hot MeOH (10 mL) to give the title compound (3.9 g, 66%)as an off-white solid.

EXAMPLE 18 Preparation of3-(5-Dimethylamino-3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-one

A solution of oxindole (938 mg, 7.05 mmol) in 20 mL of DME was cooled to0° C., and treated with 6.2 mL of 2.5M n-BuLi t hexane solution (15.5mmol) dropwise under nitrogen. The mixture was stirred at 0° C. for 10minutes. 5-dimethylaminophthalide (1.0 g, 5.64 mmol) was added as oneportion. The mixture was allowed to warm to room temperature undernitrogen and stirring was continued for 3 hours. The cloudy mixture wasthen slowly added into 0.5M aqueous HCl solution (0° C.) with vigorousstirring. The resulting mixture was neutralized with 1M NaOH solutionuntil pH=9. The yellow precipitate which formed was collected byfiltration, washed with water, dried under vacuum. The solid obtainedwas triturated with hot MeOH (20 mL), collected by filtration and driedto give title compound (990 mg, 60%) as a bright yellow powder.

EXAMPLE 19 Preparation of3-(5-Dimethylamino-3H-isobenzofuran-1-ylidene)-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-one

A mixture of3-(5-Dimethylamino-3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-one(550 mg, 1.88 mmol), paraformaldehyde (141 mg, 4.7 mmol) and piperidine(240 mg, 2.82 mmol) in 20 mL of EtOH was stirred at reflux for overnightand cooled to room temperature. The solid which formed on cooling wascollected by filtration, washed with EtOH (5 mL) and dried under vacuumto give the title compound (655 mg, 89%) as bright yellow crystals.

EXAMPLE 20 Preparation of5-Chloro-3-(5-dimethylamino-3H-isobenzofuran-1-ylidene)-1,3-dihydroindol-2-one

A solution of 5-chlorooxindole (1.18 mg, 7.05 mmol) in 20 mL of DME wascooled to 0° C., and treated with 6.2 mL of 2.5M n-BuLi/hexane solution(15.5 mmol) dropwise under nitrogen. The mixture was stirred at 0° C.for 10 minutes. 5-dimethylaminophthalide (1.0 g, 5.64 mmol) was added asone portion. The mixture was allowed to warm to room temperature undernitrogen and stirring was continued for 3 hours. The cloudy mixture wasthen slowly added into 0.5M aqueous HCl solution (0° C.) with vigorousstirring. The resulting mixture was neutralized with 1 M NaOH solutionuntil pH=9. The yellow precipitate which formed was collected byfiltration, washed with water, dried under vacuum. The solid obtainedwas triturated with hot MeOH (20 mL) followed by ethyl acetate (10 mL),collected by filtration and dried to give title compound (900 mg, 49%)as a bright yellow powder.

EXAMPLE 21 Preparation of5-Chloro-3-(5-dimethylamino-3H-isobenzofuran-1-ylidene-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-one

A mixture of5-Chloro-3-(5-dimethylamino-3H-isobenzofuran-1-ylidene)-1,3-dihydro-indol-2-one(400 mg, 1.22 mmol), paraformaldehyde (91 mg, 3.05 mmol) and piperidine(156 mg, 1.83 mmol) in 20 mL of EtOH was stirred at reflux for overnightand cooled to room temperature. The solid which formed on cooling wascollected by filtration, washed with EtOH (5 mL) and dried under vacuumto give the title compound (500 mg, 97%) as bright yellow crystals.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention only. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

What is claimed is:
 1. A compound represented by the general formula II:

wherein; A is NR²; R¹ is independently selected from the groupconsisting of halogen, hydroxy, nitro, cyano, hydrocarbyl andsubstituted hydrocarbyl radicals, wherein said substituted hydrocarbylmay be substituted with heteroatoms selected from the group consistingof halogen, nitrogen, phosphorus, sulfur and oxygen; R² is selected fromthe group consisting of hydrogen or C₁ to C₈ alkyl; R is morpholinyl; R³and R⁴, together with the nitrogen atom may form a six-memberedheterocyclic ring; R⁵ and R⁶ are independently selected from the groupconsisting of hydrogen and alkyl radicals; provided that said alkylradicals may be substituted with from one to three halo, lower alkyloxyor lower alkyl amino radicals; a is 1; b is 0 or an integer of from 1 to3; the wavy line represents a E or Z bond and Ar is phenyl.
 2. Thecompound of claim 1 wherein R⁵ and R⁶ are hydrogen.
 3. The compound ofclaim 1 wherein R³ and R⁴, together with the nitrogen atom, form a ringselected from the group consisting of morpholinyl or piperidinyl.
 4. Thecompound of claim 2 wherein R¹ is selected from the group consisting ofhalogen, C₁ to C₈ alkyl, phenyl, CF₃, OCF₃, OCF₂H, CN, SR²,(CH₂)_(d)C(O)OR², C(O)N(R²)₂, (CH₂)_(d)OR², HNC(O)R², HN—C(O)OR²,(CH₂)_(d)N(R²)₂, SO₂N(R²)₂, OP(O)(OR²)₂, OC(O)OR², OCH₂O, HN—CH═CH,—N(COR²)CH₂CH₂ HC═N—NH, N═CH—S, O(CH₂)_(d)—R⁷ and (CH₂)_(c)—R⁷ whereinR⁷ is selected from the group consisting of pyrrolidinyl, piperidinyl,pyrazinyl and morpholinyl and lower alkyl-substituted derivativesthereof; provided that R⁷ and/or said alkyl or phenyl radicals may besubstituted with from one to three, halo, hydroxyl, lower alkyloxy orlower alkyl amino radicals; c is an integer of from 1 to 2 and d is 0 oran integer of from 1 to
 5. 5. The compound of claim 1 b is
 0. 6. Thecompound of claim 1 wherein b is 1 and R¹ is chloro.
 7. The compound ofclaim 1 wherein A is —NH—.
 8. The compound of claim 1 that is selectedfrom the group consisting of3-[(4-Morpholin-4-yl-phenylamino)-methylene]-1-piperidin-1-ylmethyl-1,3-dihydro-indol-2-oneand1-Morpholin-4-ylmethyl-3-[(4-morpholin-4-yl-phenylamino)-methylene]-1,3-dihydro-indol-2-one.9. A method for treating diseases related to unregulated tyrosine kinasesignal transduction, wherein said diseases are selected from the groupconsisting of carcinoma, sarcoma, leukemia, melanoma, meningioma,glioblastoma, myoblastoma, ovarian cancer, lung cancer, colon cancer,diabetic retinopathy, age-related muscular degeneration, arthritis,restenosis, hepatic cirrhosis, atherosclerosis, surgical adhesions,glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic: microangiopathy syndrome, transplant rejection,glomerulopathies, psoriasis, diabetes mellitus, wound healing andinflammation, the method comprising the step of administering to asubject in need thereof a therapeutically effective amount of a compoundaccording to claim 1.